Assessing EGFR‐mutated NSCLC with bone metastasis: Clinical features and optimal treatment strategy

Abstract Background This study aimed to examine the clinical characteristics of bone metastasis (BoM) in patients with non‐small cell lung cancer (NSCLC) who have an epidermal growth factor receptor (EGFR) mutation and to identify the most effective treatment strategy using EGFR–tyrosine kinase inhibitors (TKIs). Methods The study included patients with stage IV EGFR‐mutated NSCLC who were receiving first‐line treatment with EGFR–TKIs between January 2014 and December 2020. These patients were divided into two groups based on the presence or absence of BoM at the time of initial diagnosis. The BoM group was further subdivided based on whether they received denosumab or not. Results The final analysis included 247 patients. Those with BoM at initial diagnosis had shorter progression‐free survival (12.6 vs. 10.5 months, p = 0.002) and overall survival (OS) (49.7 vs. 30.9 months, p = 0.002) compared to those without BoM. There was a difference in the location of metastatic sites between the two groups, with a higher incidence of extrathoracic metastasis in the BoM group (p < 0.001). The incidence of T790M was higher in patients with BoM than in those without (47.4% vs. 33.9%, p = 0.042). Multivariate Cox regression analysis revealed that sequential osimertinib treatment and the addition of antiangiogenic therapy (AAT) and denosumab therapy improved OS in patients with BoM. Conclusions The presence of BoM is a negative prognostic factor for NSCLC patients with an EGFR mutation, possibly due to the presence of extrathoracic metastases. However, adding AAT and denosumab, along with sequential osimertinib, to the treatment regimen for patients with BoM can improve survival outcomes.


Results:
The final analysis included 247 patients.Those with BoM at initial diagnosis had shorter progression-free survival (12.6 vs. 10.5 months, p = 0.002) and overall survival (OS) (49.7 vs. 30.9months, p = 0.002) compared to those without BoM.There was a difference in the location of metastatic sites between the two groups, with a higher incidence of extrathoracic metastasis in the BoM group (p < 0.001).The incidence of T790M was higher in patients with BoM than in those without (47.4% vs. 33.9%,p = 0.042).Multivariate Cox regression analysis revealed that sequential osimertinib treatment and the addition of antiangiogenic therapy (AAT) and denosumab therapy improved OS in patients with BoM.
In recent decades, lung cancer has been the world's leading cause of cancer death, despite the use of more effective treatments such as chemotherapy, immunotherapy, and targeted therapy. 1 About 15%-20% of non-Asian patients and 40%-60% of Asian patients diagnosed with non-small cell lung cancer (NSCLC) have mutations in the epidermal growth factor receptor (EGFR). 2,3Patients with advanced NSCLC with classic EGFR mutations (exon 19 deletion or L858R mutation) are treated as first-line standard drugs with EGFR-tyrosine kinase inhibitors (TKIs).This includes the first-generation EGFR-TKIs like gefitinib and erlotinib, second-generation EGFR-TKI such as afatinib and dacomitinib, as well as the third-generation EGFR-TKI, osimertinib.Initially approved for patients with acquired T790M mutations after failure on first-or second-generation EGFR-TKIs, osimertinib was later assessed as a first-line treatment due to its effectiveness against classic EGFR mutations and T790M mutations. 4Significant proportions of patients with EGFR mutation-positive NSCLC develop bone metastases (BoM), and about 40% develop it during their disease course.BoM is associated with a greater incidence of skeletal complications, lower quality of life, and a decrease in overall survival (OS). 5,6According to previous studies, it has been shown that BoM is an independent negative prognostic factor for patients with EGFR-positive NSCLC. 7BoM is not only linked to skeletal complications but also to multi-organ metastases, including brain and liver metastases, which are indicators of poor prognosis in patients with EGFR-mutated NSCLC. 8Therefore, identification of the optimal treatment strategy for these patients is urgently needed.
The available treatment options for BoM in patients with NSCLC include radiotherapy, denosumab, and zoledronic acid. 9Denosumab is a fully human monoclonal antibody with high affinity and specificity for the receptor activator of nuclear factor kappa-B ligand (RANKL).1][12] RANKL activates RANK, a member of the TNF receptor family, promoting the maturation of preosteoclasts into osteoclasts. 13,14Denosumab inhibits the interaction between RANK and RANKL, thus preventing the activation of osteoclasts and halting the vicious cycle of bone degradation. 10,15,16In a mouse model of metastatic NSCLC, suppression of RANKL reduced the burden of skeletal tumors and increased survival. 17A phase III study found that in patients with metastatic lung cancer, denosumab was associated with better OS compared to zoledronic acid. 9Additionally, a retrospective study showed that denosumab improved OS in non-squamous cell lung cancer, with a median survival of 21.4 months, compared to 12.7 months for zoledronic acid. 11imited research has been conducted on improving the prognosis of NSCLC patients with BoM who have EGFR mutations.Ihn et al. demonstrated that afatinib can suppress osteoclast differentiation induced by RANKL, reduce the expression of osteoclast-specific markers, and attenuate bone resorption. 18An in vitro study showed that osimertinib can regress tumors, improve bone remodeling, and increase survival rates in mice with BoM models, suggesting its potential as a treatment for EGFR-mutated NSCLC patients with BoM. 19A retrospective study of patients with metastatic NSCLC and EGFR mutations receiving first-line EGFR-TKI treatment revealed that combining denosumab with EGFR-TKI leads to longer OS than EGFR-TKI monotherapy. 20However, another study found that denosumab did not impact progressionfree survival (PFS) or OS. 21Recent research has explored combining EGFR-TKIs with vascular endothelial growth factor (VEGF) inhibitors in treating NSCLC patients with EGFR mutations. 22,23Some studies have observed high VEGF and receptor expression in BoM in breast or liver cancer. 24,25Chen et al. reported that antiangiogenic therapy (AAT) might improve survival chances for EGFRmutated NSCLC patients with BoM. 7 The findings underscore the importance of identifying treatment options for EGFR-mutated NSCLC patients with BoM.Consequently, this retrospective study has two objectives: (1) to compare the clinical characteristics and prognosis of EGFR-mutated NSCLC patients with and

Conclusions:
The presence of BoM is a negative prognostic factor for NSCLC patients with an EGFR mutation, possibly due to the presence of extrathoracic metastases.However, adding AAT and denosumab, along with sequential osimertinib, to the treatment regimen for patients with BoM can improve survival outcomes.

| Study design and patient population
This retrospective study was designed to investigate patients diagnosed with stage IV EGFR-mutated lung adenocarcinoma who received first-line EGFR-TKI therapy.The patient population was divided into two groups based on the presence or absence of bone metastases (BoM) at initial diagnosis, determined through bone magnetic resonance imaging (MRI), bone scan, or positron emission tomography (PET).To investigate whether denosumab improves prognosis, we further analyzed the baseline characteristics of patients who received denosumab and those who did not.Data were collected from patients treated at China Medical University Hospital between January 2014 and December 2020, excluding those with insufficient medical data, stage IIIB/IIIC disease, or patients who did not undergo re-biopsy upon disease progression.The study population includes participants from our previous study, 7 which is part of the current study.Informed consent was waived by the Institutional Review Board of China Medical University Hospital (CMUH 110-REC3-110).Baseline patient characteristics were recorded, including age, sex, smoking history, ECOG performance status, initial TNM staging, EGFR mutation subtype, metastasis patterns, first-line treatment actions, the status of the EGFR T790M mutation at disease progression (T790M mutations were identified through tissue biopsy and/or liquid biopsy), denosumab and AAT administration, and subsequent osimertinib utilization following disease progression.

| Antiangiogenesis therapy and denosumab treatment
Since bevacizumab and ramucirumab are not covered for lung cancer treatment under Taiwan's National Health Insurance program, we administered reduced dosages of these drugs in our study.Bevacizumab was given every 3 weeks at a dose of 7.5 mg/kg body weight, and ramucirumab was administered every 3 weeks at a dose ranging from 5 to 10 mg/kg body weight.Our final analysis included all participants who received a minimum of 3 cycles of combination therapy with either bevacizumab or ramucirumab.Patients in the denosumab group received a subcutaneous dose of 120 mg of denosumab once a month, starting within 1 month of BoM diagnosis and continuing for at least three treatments.During this period, they did not receive any other treatment for their BoM, including zoledronic acid.

| Assessment of outcomes
In our previous research, we discussed methods for evaluating the treatment of patients. 7To assess the baseline stage of lung cancer, we employed various imaging techniques such as computed tomography (CT), brain imaging including brain CT and/or MRI, and PET.Tumor response monitoring was conducted every 3-4 months after initiating EGFR-TKI therapy, using chest CT and clinical judgment based on brain CT or MRI.Progression-free survival (PFS) was defined as the period from the start of EGFR-TKI therapy until tumor progression, detected using the Response Evaluation Criteria in Solid Tumors v1.1, death, or censoring at the last follow-up on November 30, 2022.Similarly, OS was defined as the time between the commencement of EGFR-TKI therapy and death or censoring at the last follow-up on November 30, 2022.

| Statistical analyses
Data analysis was conducted using MedCalc for Windows, version 18.10 (MedCalc Software, Ostend, Belgium).Continuous variables with normal distributions are presented as mean ± standard deviations, while those with non-normal distributions are reported as medians and interquartile ranges.We used the t-test to determine differences between groups for continuous data with normal distributions and the Kruskal-Wallis test for non-normally distributed data.Categorical variables are expressed as numbers and percentages, with the chi-squared test used for comparing differences between independent groups.The Kaplan-Meier method was employed to evaluate PFS and OS.Univariate and multivariate Cox proportional hazards regression analyses were performed to identify independent prognostic factors of OS.A p-value <0.05 was considered statistically significant, and the strength of association was measured as a hazard ratio (HR) with an associated 95% confidence interval (CI).

| RESULTS
Between January 2014 and December 2020, 636 patients were diagnosed with EGFR-mutated stage IIIB-IV NSCLC, out of which 247 received EGFR-TKI as first-line therapy and met the inclusion criteria.These patients were divided into two groups based on the presence or absence of BoM at initial diagnosis: 126 in the BoM group and 121 in the non-BoM group (Figure 1).The baseline characteristics of the study subjects are presented in Table 1.Except for the pattern of metastasis, the demographic characteristics of the two groups were similar.Liver and adrenal metastases were more common in the BoM group than in the non-BoM group (26.2% vs. 8.3%, p = 0.002; and 18.3% vs. 5.8%, p = 0.003, respectively).However, the non-BoM group exhibited a higher incidence of pleural involvement compared to the BoM group (54.5% vs. 36.5%,p = 0.004).Notably, multi-organ metastases and extrathoracic metastases involving three or more organs were more frequent in the BoM group (p < 0.001).
After a median follow-up period of 67.4 months (range 62.2-75.7 months), 168 of the 247 patients had died.The BoM group experienced shorter PFS (10.5 months; 95% CI: 9.0-12.5 months) and OS (30.9 months; 95% CI: 25.5-35.5 months) compared to the non-BoM group (PFS: 12.6 months, 95% CI: 11.1-13.1,p = 0.002; OS: 49.7 months, 95% CI: 37.8-57.5,p < 0.001; see Figure 2A,B).The BoM group had a higher incidence of extrathoracic metastases than the non-BoM group.Among patients with extrathoracic organ metastasis, those with and without BoM accounted for 62.5% and 37.5% (p = 0.007) of the cases with one metastatic site, 80.4% and 19.6% (p < 0.001) of the cases with two metastatic sites, and 100% and 0% (p < 0.001) of the cases with three metastatic sites, respectively.This higher tumor burden in the BoM group might explain the more frequent opportunity for obtaining repeated biopsy specimens during clinical practice, possibly leading to the higher incidence of the T790M mutation compared to the non-BoM group (47.4% vs. 33.9%,p = 0.042; see Figure 3A,B).
To assess whether osimertinib could mitigate the negative impact of BoM, data on salvage therapy with osimertinib following initial treatment failure were analyzed.Among the patients who received subsequent osimertinib, 68 were in the BoM group and 60 in the non-BoM group.The BoM patients treated with subsequent osimertinib exhibited not only longer OS compared to BoM patients who did not receive osimertinib (36.5 vs. 23.3months; p < 0.001), but their OS rate was also similar to that of the non-BoM patients who did not receive subsequent osimertinib (36.5 vs. 36.6months; see Figure 4).
This study also examined the benefits of AAT and denosumab for patients in the BoM group, which was further divided based on whether patients received denosumab.Twenty patients received denosumab, 41 received AAT, and 12 received both denosumab and AAT.The group treated with denosumab had a higher proportion of patients receiving AAT compared to the group without denosumab (60% vs. 27.4%;p = 0.004; Table 2).Patients who received both AAT and denosumab exhibited an increased OS rate compared to those who did not receive these treatments (60.6 vs. 27.9months; p = 0.028; see Figure 5).Multivariate analysis was performed to test the prognostic factors identified as significant in univariate analyses, and it identified good performance status, subsequent osimertinib treatment, and combined AAT and denosumab treatment as independent positive prognostic factors for OS (Table 3).

| DISCUSSION
Our findings reveal that EGFR-mutated NSCLC patients with BoM have shorter PFS and OS compared to     Several previous studies have established that BoM is linked to a poor prognosis in EGFR-mutated NSCLC patients. 5,8,9Our study similarly found that EGFR-mutated NSCLC patients with BoM experience shorter PFS (10.5 vs. 12.6 months, p = 0.002, Figure 2A) and OS (30.9 vs. 49.7 months, p < 0.001) compared to those without.BoM is associated with multi-organ metastases, including brain and liver metastases, which are known factors of poor prognosis. 26,27The recent SEER database study indicated that lung cancer prognosis worsens with an increase in the number of metastatic sites. 28In bone metastatic cancer, the RANK/RANKL pathway plays a vital role in bone metabolism, immunity, and tumorigenesis. 29Its expression has been identified as a

F I G U R E 5
The overall survival of patients with EGFR-mutated NSCLC treated with (A) denosumab; (B) antiangiogenesis therapy; (C) denosumab and antiangiogenesis therapy.EGFR, epidermal growth factor receptor; OS, overall survival.

(A) (B) (C)
negative prognostic factor in breast cancer patients. 14he RANKL/RANK pathway is also a critical immune mediator.Recent studies have connected anti-RANKL therapy with improved responses to immunotherapy in melanoma, NSCLC, and renal cell carcinoma. 30ANKL is a negative prognostic indicator in patients with advanced KRAS-mutated lung adenocarcinoma. 31urthermore, the RANKL/RANK pathway contributes to angiogenesis and enhances vascular permeability via RANK-expressing endothelial cells, potentially impacting extravasation and metastasis processes, leading to greater tumor burden. 32Patients with BoM are more likely to develop multi-organ metastasis.Thus, we hypothesize that the RANK/RANKL pathway may be associated with poor prognosis in NSCLC patients with BoM.Denosumab has been demonstrated to have antitumor effects by blocking the interaction between RANK and RANKL. 9,11,33This inhibition suppresses the nuclear factor-kappa B pathway, which is related to lung carcinogenesis, and enhances intercellular adhesion molecule one expression and the MEK/extracellular signal-regulated kinase pathway, subsequently promoting tumor cell migration. 12The combination of denosumab and EGFR-TKI has been found to increase OS in EGFR-mutated NSCLC patients with BoM.In a retrospective study of 190 NSCLC patients with BoM, combining denosumab with first-or second-generation EGFR-TKI was associated with improved OS (26.6 vs. 20.1 months; p = 0.015). 20However, another real-world study reported that denosumab had no significant prognostic effects in lung adenocarcinoma patients with BoM treated with first-line EGFR-TKIs. 21In our study, combining EGFR-TKI with denosumab showed a nonsignificant trend toward improving the OS of EGFRmutated NSCLC patients with BoM.These inconsistent results could be attributed to factors such as insufficient sample size, variation in additional treatments at different institutions, and limited follow-up duration.
An animal study on osteoclasts demonstrated that the ERK1/2 pathway regulates osteoclast invasion induced by RANKL and VEGF. 34VEGF is involved in regulating RANK/ RANKL-mediated osteoclastogenesis. 35The immune modulatory effects of antiangiogenic agents targeting the VEGF/ VEGF receptor on various immune cells, including effector T cells, Tregs, myeloid-derived suppressor cells, dendritic cells, tumor-associated macrophages (TAMs), and mast cells, in the tumor microenvironment of NSCLC, have been well-documented. 36Additionally, the RANK/RANKL pathway is linked to resistance to immunotherapy, as RANKL attracts TAMs and RANKL/RANK signaling in M2 macrophages promotes Treg lymphocyte proliferation, creating an immunosuppressive environment. 37There is potential crosstalk between the RANK/RANKL and VEGF pathways in the metastatic bone and tumor microenvironment of NSCLC.A systematic review and meta-analysis showed that combining EGFR TK inhibitors and anti-VEGF agents in patients with EGFR-mutated NSCLC could improve PFS. 38ur previous study indicated that EGFR-mutated NSCLC patients with BoM could benefit in survival by receiving additional anti-VEGF treatment. 7In the current study, we found that the combination therapy of EGFR-TKI, denosumab, and an anti-VEGF agent significantly improved OS compared to EGFR-TKI monotherapy in patients with EGFR-mutated NSCLC.The combination therapy group had a longer OS of 60.6 months versus 27.9 months in the monotherapy group (p = 0.028).Moreover, after multivariate analysis, the combination of denosumab and an anti-VEGF agent was identified as a prognostic factor, with an odds ratio of 0.23 (95% CI: 0.06-0.87,p = 0.031).
Osimertinib, a third-generation EGFR-TKI, is extensively used to treat NSCLCs that harbor the T790M mutation and is an effective sequential therapy for EGFR-mutated NSCLC patients with an acquired T790M mutation after first-or second-generation EGFR-TKI treatment. 39It is also preferred as a first-line therapy over first-generation EGFR-TKI drugs. 40 erlotinib (n = 183), osimertinib (n = 150), or afatinib (n = 55) found that osimertinib significantly prolonged PFS in patients with BoM and the exon 19 deletion, compared to other EGFR-TKIs. 41An in vitro study showed that osimertinib induced tumor regression, improved survival in mice, and caused bone remodeling in bone metastatic models. 19The present study identified sequential therapy with osimertinib as a prognostic factor for EGFRmutated NSCLC patients with BoM.Our findings indicate that EGFR-mutated NSCLC patients with BoM receiving first-or second-generation EGFR-TKI are more likely to develop T790M resistance after TKI failure (47.4% vs. 33.9%,p = 0.042).This could be another factor contributing to the improved OS observed with osimertinib sequential therapy.The present study has several limitations.First, its retrospective design includes only a limited number of patients who received denosumab or anti-VEGF treatment.Despite denosumab being commonly used in Taiwan following a reimbursement program initiated by National Health Insurance on December 1, 2015, not all physicians prescribe it for managing bone diseases or skeletal-related events (SREs) in NSCLC patients. 6,20,21dditionally, anti-VEGF medicine is not covered by Taiwan Health Insurance.Therefore, large-scale clinical trials are necessary to conclusively demonstrate the benefits of combining denosumab and anti-VEGF with EGFR-TKIs.Second, in real-world scenarios, the start time for denosumab varies, complicating the assessment of its impact on PFS.In our study, OS was the primary indicator when combining denosumab with EGFR-TKI treatment.Third, our study does not report on SREs, which previous studies have identified as a poor prognostic factor. 20,21Fourth, we used different dosages of bevacizumab (7.5 mg/kg every 3 weeks) and intravenous ramucirumab (5-10 mg/kg every 3 weeks) compared to prior trials, 22,23 due to these drugs not being covered for lung cancer treatment under Taiwan's National Health Insurance, leading to the use of reduced dosages.Previous research showed that combining chemotherapy with 7.5 mg/kg of bevacizumab was as effective as using 15 mg/kg. 42Finally, this study mainly involved patients receiving first or second-generation EGFR-TKIs as first-line therapy.The global shift to using osimertinib as a first-line treatment may influence our findings.However, the optimal sequencing of these TKIs is crucial for survival outcomes.Given the limited treatment options post-osimertinib, subsequent osimertinib therapy after first or second-generation EGFR-TKI failure remains a feasible strategy.Despite these limitations, our study is the first to explore combination therapy with denosumab and AAT for EGFR-mutated NSCLC patients with BoM.

| CONCLUSION
Our study indicates that BoM is common in EGFRmutated NSCLC patients and is associated with a poor prognosis.For those experiencing EGFR-TKI failure and having BoM, osimertinib is a preferred option for sequential therapy due to the high incidence of the T790M mutation.The combination of an EGFR-TKI with either denosumab, an anti-VEGF agent, or both, has been shown to improve OS in these patients.Specifically, the hybrid combination of EGFR-TKI, denosumab, and an anti-VEGF agent appears to have a synergistic effect on OS.Further research is necessary to confirm the synergistic impact of combining denosumab and anti-VEGF agents.

F I G U R E 1
Flowchart showing patient selection.EGFR, epidermal growth factor receptor; NSCLC, nonsmall-cell lung cancer; PD, progressive disease; TKI, tyrosine kinase inhibitor.

| 5 of 11 CHEN
et al. those without BoM.Additionally, patients with BoM are more likely to develop metastases in multiple organs.After initial treatment failure, osimertinib appears to be a preferable choice for sequential therapy, especially given the high incidence of the T790M mutation in EGFR-mutated NSCLC patients with BoM.The triple

F
I G U R E 2 (A) PFS of patients treated for EGFR-mutated NSCLC with or without bone metastasis (BoM).(B) OS of patients treated for EGFR-mutated NSCLC with or without BoM.PFS, progression-free survival; OS, overall survival.combinationtherapy of EGFR-TKI, denosumab, and an anti-VEGF agent can synergistically improve OS in these patients.To our knowledge, our study is the first to investigate the survival benefits of combining AAT and denosumab for EGFR-mutated NSCLC patients with BoM.

F I G U R E 3 F I G U R E 4
(A) The proportions of EGFR-mutated NSCLC patients with or without bone metastasis (BoM) with extrathoracic metastasis.(B) The incidence of T790M in EGFR-mutated NSCLC patients with or without BoM after 1st line treatment failure.EGFR, epidermal growth factor receptor; PD, progressive disease.The overall survival (OS) of EGFR-mutated NSCLC patients with or without bone metastasis who received osimertinib treatment and those who did not.EGFR, epidermal growth factor receptor; NSCLC, non-small-cell lung cancer.
The baseline clinical characteristics of EGFR-mutated NSCLC patients with or without bone metastasis (BoM).
T A B L E 1Abbreviations: CNS, central nerve system; ECOG, Eastern Cooperative Oncology Group; EGFR, epithelial growth factor receptor; NSCLC, non-small-cell lung cancer.
The baseline clinical characteristics of EGFR-mutated NSCLC patients with bone metastasis (BoM) who received or did not receive denosumab.
T A B L E 2Abbreviations: CNS, central nerve system; ECOG, Eastern Cooperative Oncology Group; EGFR, epithelial growth factor receptor; NSCLC, non-small-cell lung cancer.
A retrospective study involving advanced NSCLC patients receiving gefitinib/ Cox proportional hazards regression analysis of the OS of patients with EGFR-mutant NSCLC with bone metastasis (BoM).